1. Field of the Invention
This invention relates generally to techniques used in molecular biology. In particular, it relates to an improved reaction mixture for use in in vitro RNA trancription and in various other enzymatic reactions in which a polynucleotide is synthesised. The reaction mixture uses high concentrations of total nucleotides, at levels that were previously thought to be inhibitory; a concentration of Mg++ that is subsaturating with respect to the nucleotide concentration, the enzyme inorganic pyrophosphatase, and most preferrably, also Mg++- or Tris-nucleotides.
2. Description of the Related Art
The techniques used in the field of molecular biology have been widely and successfully applied, not only in many areas of basic research, but also in providing solutions to several medical and agricultural problems. As such, molecular biology is of great importance to scientific research, medicine, human welfare and the economy.
RNA and DNA polymerization reactions, which result in the synthesis of RNA or DNA polynucleotides, are an integral part of a variety of techniques used in molecular biology. Obviously, an increase in the yield of these reactions would be beneficial, both in saving time and expense. Such reactions include, in vitro transcription reactions, amplification techniques such as the polymerase chain reaction (PCR), self-sustained sequence replication (3SR), QB replicase and others. These reactions often employ bacteriophage RNA polymerases, such as SP6, T7 and T3, for example, in the synthesis of both radiolabeled RNA probes and unlabeled RNA.
The rate of these synthetic reactions, and the amount of product formed, is known to be limited by several factors. A common belief in the art is that these limiting factors cannot generally be overcome, and that the yield of these reactions cannot be significantly increased. For example, it is thought that nucleotide concentrations greater than 8 mM are inhibitory to in vitro transcription reactions (Gurevich et al., 1991).
In principle, the yield of reactions can generally be increased by increasing the levels of substrate(s), in this case the concentration of nucleotides used in the reaction. However, in the synthesis of polynucleotides, high levels of the nucleotide substrate are known to act as competitive inhibitors of the polymerization reaction and actually decrease the yield of the reaction. They can also lead to increases in the error rate, which is highly undesirable. The Km for the nucleotide substrates are quite low (ATP, 47 xcexcM; GTP 160 xcexcM; UTP, 60 xcexcM; CTP 81 xcexcM) (Chamberlin and Ring, 1973). Thus, nucleotide concentrations in the mM range are likely to be saturating, and, as mentioned above, total nucleotide concentrations above 8 mM have been reported to inhibit RNA synthesis (Gurevich et al., 1991). It is likely that this inhibition is due to competitive inhibition of the polymerase by the high level of nucleotides. This phenomenon is discussed by Chamberlin and Rhodes (1974), in regard to E. coli RNA polymerase.
Considering the inhibitory effects that are believed to result from using high nucleotide concentrations, the total nucleotide concentrations currently used for reportedly optimal RNA synthesis range from 1.6-16 mM (Milligan et al., 1987; Sampson and Uhlenbeck, 1988; Cunningham and Ofengand, 1990; Weitzmann, et al., 1990; Gurevich et al., 1991; Wyatt et al., 1991). It is particularly noteworthy that xe2x80x98Current Protocols in Molecular Biologyxe2x80x99, a standard laboratory manual, recommends the lowest total nucleotide concentration, of 1.6 mM.
The substrate for polynucleotide synthetic reactions is actually a complex of the nucleotide with magnesium ions, i.e. a Mg++-nucleotide (Mg++-NTP), and therefore the magnesium concentration is also an important parameter. The conventional procedure is to routinely add magnesium at a concentration greater than the total nucleotide concentration. Indeed, the current view is that an excess of Mg++ must be added in in vitro transcription reactions (Milligan and Uhlenbeck, 1989). It also been reported that a ratio of magnesium to total nucleotides of 1.75:1 is optimal (Wyatt et al., 1991).
The enzyme inorganic pyrophosphatase has recently been used in transcription reactions (Sampson and Uhlenbeck, 1988; Weitzmann et al., 1990; Cunningham and Ofengand, 1990), and DNA polymerase and DNA sequencing applications (Tabor and Richardson, 1990). The Cunningham and Ofengand study concluded that the pyrophosphatase increases transcription yields and also minimizes the effect of variation of magnesium concentration, although this was not believed to be due to the pyrophosphate sequestering the Mg++.
A further complication in polynucleotide synthesis is the fact that most phage polymerases have a low salt optima, SP6 polymerase in particular is very sensitive to even low levels of Na+ (Butler and Chamberlin, 1982). Despite this, in most reactions of this nature nucleotide-salts are used, and in particular, Na+-nucleotides. Indeed, most commercially available nucleotides are the Na+, Li+, K+, NH4+ or Ba++ salts. Increasing the levels of these nucleotides above those generally used would result in the introduction of amounts of salt which are inhibitory to the reaction.
The present invention seeks to overcome some of these and other drawbacks in the prior art by providing improved compositions for in vitro RNA trancription and various other enzymatic reactions in which a polynucleotide is synthesised. In particular, an improved reaction mixture is disclosed comprising high concentrations of total nucleotides, in the order of between about 12 mM and 40 mM, that were previously thought to be inhibitory; an effective molar concentration of Mg++ that is subsaturating with respect to the molar total nucleotide concentration, the enzyme inorganic pyrophosphatase, and most preferrably, also Mg++- or Tris-nucleotides. The invention further relates to methods for employing this reaction mixture in various synthetic procedures.
As used herein, the term total nucleotide concentration is intended to refer to the total concentration of nucleotides (NTPs), i.e. the sum of the concentrations of ATP, GTP, CTP, and/or UTP, present initially in the reaction mixture when the various components of the reaction mixture have been assembled in the final volume for carrying out the reaction. Naturally, as the reaction proceeds, the nucleotides themselves will be incorporated into the polymer and so the concentration of total nucleotides will be progressively reduced from its initial value.
The process of synthesizing a polynucleotide is subject to substrate inhibition by the magnesium complexes of the various nucleotides, Mg++-NTPs. Higher concentrations of Mg++-NTPs slow the rate of polymerization as an excess of each of the other species inhibits the incorporation of the correctly matched nucleotide into the growing polymer. An important aspect of the present invention is to provide a means of increasing the total nucleotide concentration without inhibiting the reaction.
The present invention provides a reaction mixture in which the total nucleotide concentration is greater than 12 mM and not so high as to substantially inhibit the reaction to be performed. It is proposed that for most applications the upper limit nucleotide concentration will be about 100 mM, however, a higher upper limit may be appropriate under certain circumstances. For most applications, the nucleotide concentration will be between 12 mM and about 40 mM. In preferred embodiments, the total nucleotide concentration will be between about 16 mM and about 40 mM, and more preferrably, between about 20 mM and about 40 mM.
The inventors have provided other components for use in the present reaction mixture, and further adjusted the concentrations of the various constituents, such that these higher total nucleotide concentrations do not inhibit the reaction as one may expect, but in fact act to stimulate the total amount of product synthesized and possibly the rate of polymerization.
As the substrate for the polymerization reaction is the Mg++-nucleotide complex, an effective concentration of Mg++ is herein defined as one that will form a complex with the nucleotides in the reaction mixture to allow the polymerization reaction to procede.
The present inventors have determined that the use of effective Mg++ concentrations that are subsaturating with respect to the total molar nucleotide concentration is particularly advantageous. To create a reaction mixture in accordance with the present invention in which the Mg++ concentration is subsaturating with respect to the total nucleotide concentration, it is not necessary for the absolute Mg++ concentration to be less than the total nucleotide concentration. The reason for this is that not all the Mg++ ions in the reaction mixture will be available to form a Mg++-NTP complex. For example, some of the Mg++ ions may be sequestered by inorganic pyrophosphate, depending on its generation in the reaction. Therefore, a Mg++ concentration that is subsaturating with respect to the total molar nucleotide concentration is herein defined as one that is not more than 10% greater than the total nucleotide concentration. However, in preferred embodiments, it is contemplated that the Mg++ concentration used will be equal to the total molar nucleotide concentration, and even more preferrably, that it will be less than the total molar nucleotide concentration.
Another parameter for assessing the Mg++ in the reaction is to calculate the xe2x80x9cfreexe2x80x9d Mg++, as opposed to the total Mg++. Naturally, in any solution, the xe2x80x9cfreexe2x80x9d concentration of an ion will always be less than the total concentration. In this case, the total Mg++ is all the Mg++ introduced into the reaction, while free Mg++ is that Mg++ not bound to nucleotides, enzymes and other constituents in the reaction mixture. The free Mg++ will therefore be dependent on the other constituents in the reaction mixture. To calculate the free Mg++ in the reaction mixture, one would use a software program, such as Maxchelate, version 4.12, (Chris Patton, Hopkins Marine Station, Stanford University, Pacific Grove, Calif. 93950). This program uses the association constants for Mg++ and nucleotides and takes into account the pH of the reaction which will affect the association constant. The inventors have used this program to calculate the free Mg++ for the experiment shown in FIG. 2. The values for free Mg++ are shown in Table 1 below. An example of this is a calculation that compares the free Mg++ for the study shown in FIG. 2 to that of a study disclosed by Cunningham and Ofengard (1990).
As such, in certain embodiments, the present invention concerns a reaction mixture for the synthesis of a polynucleotide in which the xe2x80x9cfreexe2x80x9d Mg++ concentration is equal to or less than 2xc3x9710xe2x88x924M.
A further important aspect of the present invention is the inclusion within the reaction mixture of the enzyme inorganic pyrophosphatase, which catalyzes the hydrolysis of inorganic pyrophosphate. Being a product of the reaction, inorganic pyrophosphate may function as an end product inhibitor to limit the rate of the polymerization. The addition of pyrophosphatase to the reaction mixture allows pyrophosphate to be removed and thus prevents its direct inhibitory action. Furthermore, the inventors believe that the pyrophosphate molecule sequesters the Mg++ ions and prevents them from forming the concentrations of Mg++-NTP substrate complexes which are inhibitory to the polymerase in the early stages of the reaction. However, later in the reaction, when the nucleotide levels have been depleted, removing pyrophosphate serves to free Mg++ and promote Mg++-NTP formation and thus allows polymer synthesis to occur with subsaturating levels of Mg++.
Levels of inorganic pyrophosphatase: The concentration of inorganic pyrophosphatase in the reaction mixture is not believed to be particularly critical, so long as it is sufficient to catalyze the hydrolysis of pyrophosphate. The inventors contemplate that an amount of pyrophosphatase corresponding to between about 10 and about 50 international units (U)/ml of reaction mixture of enzyme activity may be used, and more preferrably, an amount corresponding to approximately 15 U/ml. One international unit of pyrophosphatase activity is defined as the amount of enzyme that will liberate 1.0 xcexcM of inorganic orthophosphate per minute at pH 7.2 at 25xc2x0 C.
A further aspect of the present invention relates to the form in which the nucleotides are added to the reaction mixture. The inventors contemplated that the use of nucleotides in the form of a salt with Na+, K+, Ba++, or NH4+, as commonly-used, may lead to the inhibition of the polymerization reaction by the counter ions. Therefore, in certain embodiments, it is proposed that the nucleotides may be added to the reaction mixture in a form other than as a compound with Na+, K+, Ba++, or NH4+, such as a Mg ++-nucleotide or a Tris-nucleotide. The use of Mg++- or Tris-[Tris(hydroxymethyl)aminomethane] nucleotides is thought to be particulary advantageous when using SP6 polymerase. However, the present invention is not limited solely to the use of Mg++- or Tris-nucleotides, as certain advantages will result from the use of the above-described reaction mixture irrespective of the form in which the nucleotides are added. For example, many basic compounds will be suitable counter ions for nucleotides. Possible examples include glycylglycine, Tricine or Bicine.
Of course, other components in addition to those described above will need to be added to the reaction mixture in order to achieve polynucleotide synthesis. One will wish to provide a buffered solution of a suitable ionic strength and pH and a reducing agent, such as 40 mM Tris-HCl, 10 mM dithiothreitol (DTT), 2 mM spermidine-HCl, pH 8.0. Naturally, one will ultimately need to include a polymerase enzyme and a linearised polynucleotide template for the polymerase to use in synthesizing the complementary nucleotide strand. It is also considered to be advantageous to include a component to limit the degradation of the polynucletide, such as a ribonuclease inhibitor.
The reaction mixture of the present invention will find particular utility in in vitro RNA transcription reactions. However, in certain embodiments, the present invention also relates to the preparation of both polyribonucleotides and polydeoxyribonucleotides using other methods. In the former case, the enzyme used in conjuction with the reaction mixture will be an RNA polymerase, and in the latter, it will a DNA polymerase. The invention is not limited to the use of any particular polymerase enzyme. Indeed, it is contemplated that any enzyme, either of bacteriophage- or non phage-origin, that catalyzes the formation of a polynucleotide will be suitable. However, certain RNA or DNA polymerases are contemplated to be of particular use in accordance with the present invention. For example, RNA polymerases such as T7, or T3, or SP6 RNA polymerase; and DNA polymerases such as Taq, or T7 or T4 polymerase, or Klenow polymerase.
The present inventors have discovered that the use of higher concentrations of total nucleotides in conjunction with the above-described reaction mixture results in unexpectedly large increases in the formation of a polynucleotide. For example, standard yields achieved in RNA synthesis are generally on the order of 10-20 moles of product per mole of template (Krieg and Melton, 1987). Significantly higher yields have been reported by other workers. For example, Cunningham and Ofengand, (1990) reported 627 moles of product per mole of template using T7 RNA polymerase. It is difficult, however, to directly compare yields from different laboratories because the different templates used have different intrinsic transcriptional efficiencies. Using T7 RNA polymerase, the present inventors compared the transcriptional yields using the conditions described by Cunningham and Ofengand, (1990) and our conditions described in the legend to FIG. 2. After a 6 hour incubation, the yield with the Cunningham and Ofengand, (1990) conditions was 196 moles of product per mole of template. With our best conditions, the yield was 561 moles of product per mole of template, which represents more than a 2.5 fold improvement. This experiment is shown in Table 3. Thus, the creation of conditions which allow nucleotide concentrations that were previously thought to be inhibitory to be used successfully has yielded unexpected benefits.
In certain embodiments, the invention relates to methods of preparing a polynucleotide using the reaction mixture of the present invention. As mentioned above, the reaction mixture is believed to be particularly suitable for use in in vitro RNA transcription reactions. To synthesize an RNA molecule in this manner, one would prepare a reaction mixture as described above, add to it a polynucleotide template and an RNA polymerase enzyme, and incubate the reaction mixture. Either radioactively labeled polynucleotides, for use as probes, or unlabeled polynucleotides may be prepared, by using labeled or unlabeled substrate nucleotides.
In further methodological embodiments, the above-described reaction mixture may be used in other polynucleotide synthetic reactions. Methods of this sort are intergral parts of several commonly-used techniques in molecular biology apart from in vitro RNA transcription, these include amplification techniques such as polymerase chain reaction (PCR), self-sustained sequence replication (3SR), QB replicase and others. In each of these techniques, increases in the yield of the reaction would be beneficial. It is proposed that the present invention provides a means that this may be achieved by using the reaction mixtures disclosed herein. Furthermore, the invention is not limited to reactions in which a polymerase enzyme is used. For example, other techniques that could benefit from using the present reaction mixture include the DNA and RNA ligase reactions in which ATP is hydrolysed to AMP and pyrophosphate.